Effective tissue fabrication depends on the incorporation of an integrated vascular system into the tissue construct. The vasculature itself is a complex, multi-cellular system with unique but different biological requirements. At the single vessel level, the general structure entails a tube of which the walls are comprised of different cellular layers, each of which impart structural and functional characteristics to the vessel. However, a single vessel will contribute effectively to tissue perfusion only when incorporated into a vascular network. Any one vascular network entails in-flow and outflow vessels (arteries and veins) delivering and draining blood to and from the downstream distribution and tissue-interface vessels (the microvasculature).
The ability to establish and maintain a functional, adaptable microcirculation in vitro has the potential to significantly impact a broad array of biomedical arenas. In virtually every discussion concerning the building of tissue replacements, the critical importance of having a microvasculature integrated into the tissue construct is stressed. In cellular assay platforms, the presence of a perfused vasculature in combination with the target parenchyma cell is considered to improve the utility of the assay beyond having just parenchyma cells. An in vitro perfused microvasculature model with a functional, truly adaptive microcirculation would permit investigation of the mechanisms of microvascular form and function, such as how flow affects angiogenesis, and those mechanisms underlying microvessel wall and network remodeling. Drug discovery, investigation of vascular disease mechanics, and environmental chemical toxicity, are all areas with the potential to be benefited by development of an in vitro, perfusable microvasculature that fully recapitulates physiological microcirculation function and adaptability.
Formally, a microcirculation is a system of blood circulation through the microvasculature. Importantly, a microvasculature capable of supporting effective circulation consists of a properly arranged hierarchical tree of heterogeneous vessel types producing a network topology forming inflow (arterioles), exchange (capillaries), and outflow (venules) pathways. The individual microvessels that make up a network are complex structures made up of not only endothelial cells but also a variety of mural cells, all of which are necessary to form a stable and adaptable microvessel. The ability of the entire network to adapt and appropriately remodel in response to a variety of hemodynamic cues is a necessary homeostatic activity intrinsic to all microvasculatures, and is thought to reflect, in part, an effort by the vessels to normalize hemodynamic forces (herein called “resistance-based adaptation”. This ability is an important determinant of endothelial cell health and function. Thus, an ideal in vitro microcirculation is a perfused microvasculature that is comprised of all of the vascular cells required for recapitulating these core microvascular activities.
A key goal of current innovation is therefore generation of a truly adaptable functional in vitro microcirculation. A network of human vessels is required to create and maintain 3-D tissues of physiologically relevant proportions. One early strategy for creating vascular-like perfusion circuits involved lining pre-formed channels of microvessel-like dimensions (usually 6-200 μm in diameter) with vascular cells. A common approach to forming these “microchannels” utilized standard soft lithography to mold vascular-compatible matrices into 2-D or 3-D channels which are subsequently lined with endothelial cells (and sometimes mural cells). This general strategy has been successfully used to form predominately parallel, endothelialized channels as part of a microfluidic-based “vascularized” system. In many respects, however, the “vessels” formed are merely lined walls of channels with fixed microvascular dimensions in a fixed network topology. (See, e.g. U.S. Pat. No. 8,663,625 to Stroock et al.) Consequently, while useful in applications benefiting from an endothelial cell-lined perfusion circuit (e.g. such as in biochip applications), such vascularized systems are limited in their ability adapt to hemodynamic changes and parenchyma requirements.
Recently, a team of investigators purported to have developed a “living and dynamic” in vitro perfused human capillary network that is metabolically responsive and adaptable. (U.S. Ser. No. 13/253,820, titled “High-Throughput Platform Comprising Microtissues Perfused With Living Microvessels” to George et al. published Apr. 5, 2012, and Tissue Engineering: Part C Vol. 19, 2013 p 1-8, collectively referred to herein as “George”.) According to George, the system provides matrix, cells and angiogenic stimuli that allow “capillaries’ to self assemble into a continuous network and subsequently anastomose with adjacent fluidic channels to form a “living dynamic” in vitro microcirculation perfusable at physiological flow and shear rates. The in vitro microcirculation of George, however, is not a native, bona fide microcirculation. The George lab utilized cultured, single cells that assembled into cellular tubes that connected the microfluidic channels to each other through the seeded microtissue. George refers to the cellular tubes as “vessels,” and has demonstrated that the tubes re-orient and inosculate. To this extent they are not unlike capillaries, however do not have the complex, multicellular, multi-laminate structure of native arterioles or venules and do not, therefore, exhibit appropriate resistance-based adaptation nor true microvessel function.
“Microcirculation” is generally taken morphologically, to encompass all of the blood vessels with a diameter of less than 150 μm, that is, some small arteries, arterioles, capillaries, and venules. The complexity and hemodynamic response of each type of vessel is unique. Capillaries are generally the most simple vessels, ranging from 4 to 12 μm in diameter of which the walls are composed exclusively of endothelial cells, each endothelial cell, rolled up in the form of a tube and composing one segment of the capillary. Arterioles and venules, on the other hand, are multilaminate, complex, and arterioles in particular include a thicker myogenic layer that is largely responsible for resistance-based adaptive remodeling. Each type of vessel responds differently to flow hemodynamics and a native microcirculation includes a network of microvessels composed necessarily of all three types. An in vitro microcirculation must at least recapitulate these functionalities in order to be truly adaptive. At the very most, the protocol of George results in a network of immature capillaries.
Established microcirculation tenets say that an immature, dysfunctional microvessel network will eventually resolve down to a single, large caliber microvessel since the elements necessary to establish proper network architecture/topology are not in place. Persons of skill in the microcirculation arts commonly refer to this as a “shunt problem” (described and exemplified in Pries, et al. The shunt problem: control of functional shunting in normal and tumor vasculature. Nature Reviews Vol. 10, August 2010, pp 587-593).
Interestingly, this reductive disappearance of microvasculature in favor of a single larger tube was exhibited by the George “microcirculation” in response to physiological flow parameters and was presented as evidence that the in vitro “microcirculation” was capable of adaptation. A normal part of microcirculatory adaptation is indeed the capacity to delete extraneous neovessels from a network. Too much of this “pruning,” however, leads to microvessel rarefaction (disappearance of the capillary bed) and reflects a dysfunctional microcirculation. The neovasculature of George did not include vessels having a native mural layer, which is the myogenic layer responsible for adapting the vessel morphology/diameter in response to changing hemodynamic forces. In the absence of this capacity, the vessel is damaged and pruning is triggered. Microcirculation adaptability and remodeling is a dynamic process dependent on a complex and not completely understood interplay of various growth factors, hemodynamic forces, and vessel health status. Adaptive, positive, outward remodeling is a reactive and compensatory response to stimuli and stress. Maladaptive negative-inward constrictive remodeling eventually results in narrowing and disappearance of microvessels, and the resulting microcirculation rarefaction. Although George is technically correct in characterizing the observed rarefaction as an adaptation, a person skilled in the art of microcirculation recognizes that this adaptation is inappropriate and reflects a dysfunctional, unstable in vitro “microcirculation” of limited investigative utility.
Recapitulating a truly functional physiological neovascularization requires more than the generation of new vessel elements. Physiological neovascularization requires vascular guidance and inosculation, vessel maturation, pruning, A-V specification, network patterning, structural adaptation, intussusception, and microvascular stabilization. Without the concomitant capacity for neovessel remodeling and adaptation, networks of simple, non-specialized vessel segments give rise to a dysfunctional microcirculation.
In the case of in vivo engineering of microcirculations for implantation, nearly two decades of research has established that incorporation of endothelial cells alone (particularly human endothelial cells) into a tissue scaffold does not effectively result in formation of a stable microvasculature once implanted. The presence of additional perivascular cells or precursors, such as smooth muscle cells, mesenchymal smooth muscle precursors (e.g. 10T1/2 cells), and/or tissue stromal cells in the engineered system promotes neovascularization and is needed for long-term microvascular stability. It was previously established that new stable microvessel segments may be integrated into a microfluidic network via angiogenesis (formation of new vessels from existing vessels) and neovascularization (formation of a circulatory network). This is perhaps best highlighted in the use of isolated, intact microvessel segments, which retain the native microvessel structure (Hoying et al. Angiogenic potential of microvessel fragments established in three-dimensional collagen gels. In Vitro Cell Dev Biol Anim. 1996; 32:409-419, the entire disclosure of which is incorporated herein by this reference), to rapidly form a mature, functional microcirculation in vivo. Hoying and his colleagues later developed and characterized an experimental model of tissue vascularization based on the implantation of this microvascular construct, which was shown to rapidly inosculate with recipient host circulation and to recapitulate physiological angiogenesis, vessel differentiation, and network maturation (Hoying et al. Rapid Perfusion and Network Remodeling in a Microvascular Construct after Implantation, Arterioscler Thromb Vasc Biol. 2004 May; 24(5):898-904, the entire disclosure of which is incorporated herein by this reference). While the perivascular cells in these composite vascular tissue constructs play multiple roles related to neovascularization, an important function of these cells is to maintain neovessel stability.
Creating a microenvironment that enables growth of an in vitro microtissue perfused with living microvessels (e.g., arterioles, capillaries, and venules) represents a completely new paradigm. By definition, a 3-D tissue requires enhanced transport of nutrients and waste relative to 2-D monolayer cultures. Current approaches to create such an environment include: 1) enhanced concentration gradients of nutrients and waste while relying on molecular diffusion as the mode of transport, 2) creation of microchannels in the tissue to enhance advection (forced convection), and 3) forced interstitial fluid flow. In vivo, diffusion of nutrients and waste is the mechanism of transport once solutes exit the capillary bed, and is generally limited to distances <250 μm. The rate of transport is proportional to the concentration difference between two points, and inversely related to the separation distance. Hence, numerous 3-D tissue models have been reported with dimensions on the order of 1-10 mm by simply enhancing the oxygen tension (room air is 160 mmHg compared to 20-30 mmHg in the interstitial tissue) and concentration of other nutrients (e.g., glucose). Clearly, development of a truly functional, adaptive microcirculation is an important step in the evolution of 3-D tissue and organ fabrication technology.
Thus, although the ability to generate living adaptable microvessels in 3-D networks that become functional upon implantation has been demonstrated, development of a stable and adaptable in vitro microcirculation has not heretofore been achieved.
There remains a need for an in vitro perfusion device vascularized with a recapitulated physiological microcirculation that is stable and appropriately adaptable and which may be subjected to stimuli/putative agents/forces via perfusion or environmental/nutritional manipulation for a variety of downstream applications and for continued investigative utility.